Thiosulfates

In 2015, fifty different biparental Crassostrea gigas oyster families were produced from wild seed broodstocks sampled in farming and non-farming areas in two geographic regions (French Mediterranean and Atlantic coasts, Supplementary Table 1). In the Atlantic area, 73 oysters were collected at Logonna Daoulas (farming area) and 70 oysters at Dellec (non-farming area). In the Mediterranean area, 125 oysters were collected in the Thau lagoon (farming area) and 65 at the Vidourle river mouth (non-farming area). In addition, 84 oysters issued from a mass selection programme to enhance their resistance to mortality syndrome were used^{28 (link)}. All the collected oysters were transferred to the Ifremer facility at Argenton (Brittany, France) between 6 and 8 January 2015 and treated for 6 days with chloramphenicol (8 mg/l).
For gametogenesis induction, animals were held for 8 weeks in 500 l flow-through tanks with seawater enriched with a phytoplankton mixture at a constant temperature of 17 °C^{13 (link),22 (link)}. Seawater was UV-treated and filtered through 10-μm mesh. The daily mixed diet consisted of Tisochrysis lutea (CCAP 927/14; 40 μm³, 12 pg cell⁻¹) and Chaetoceros muelleri (CCAP 1010/3; 80 μm³, 25 pg cell⁻¹). Once the oysters were reproductively mature, gametes from 91 individuals (46 males, 45 females) were obtained by stripping. Gametes from one male and one female from the same origin were mixed in a 5-l cylinder at a ratio of 50 spermatozoids per oocyte (day 0). The fertilized oocytes completed their embryonic development in 5-l tubes filled with 1-μm-filtered, UV-treated seawater at 21 °C for 48 h. The D-larvae (day 2) were then collected and reared in flow-through rearing systems at 25 °C^{45 (link)}. At the end of the pelagic phase (day 15), all the larvae were collected on a 100-μm sieve and allowed to settle on cultch. Post-larvae were maintained in downwelling systems, where they were continuously supplied with enriched seawater until the experiments began. In the larval and post-larval stages, the oysters were fed the same diet as the broodstock at a concentration between 1500–2000 μm³ μl^{−145 (link)}.
Of the 50 families of oyster seed produced, 3 families from each location were kept, along with 3 other families from the mass selected broodstock, for ‘natural’ experimental infections. These 15 oyster families were maintained under highly controlled biosecured conditions to be sure that no oyster pathogens would interfere with further experiments. The ‘pathogen-free’ status of the animals was confirmed by (i) the absence of OsHV-1 DNA detection by qPCR and (ii) a low Vibrio presence (~10 cfu⁻¹ tissue) determined by isolation on selective culture medium (thiosulfate-citrate-bile salts-sucrose agar, TCBS)^{12 (link)}. Oysters were observed to remain free of any abnormal mortality throughout the larvae until the beginning of the ‘natural’ experimental infections.

For the isolation of V. cholerae, 500 ml of water was collected in a sterile 500-mL Nalgene (http://nalgene.com/) bottle from each fixed site; the samples were transported at ambient temperature to the University of Florida laboratory at Gressier and processed for detection of V. cholerae within 3 hours of collection.
In addition to the conventional sample enrichment technique (18 (link)), we used alkaline peptone water (APW) to enrich water samples. A 1.5-mL water sample was enriched with 1.5 mL of 2× APW in 3 tubes: 1 tube was incubated at 37°C for 6–8 hours (18 (link)), another tube was incubated overnight at 37°C, and the third tube was incubated at 40°C for 6–8 hours. Subsequently, a loopful of culture from each tube was streaked onto thiosulfate citrate bile salts sucrose agar (Becton-Dickinson, Franklin Lakes, NJ, USA), and the plates were incubated overnight at 37°C. From each plate, 6–8 yellow colonies exhibiting diverse morphology were transferred to L-agar; these plates were incubated overnight at 37°C. Each colony was examined by using the oxidase test; oxidase-positive colonies were tested by using V. cholerae O1–specific polyvalent antiserum and O139-specific antiserum (DENKA SEIKEN Co., Ltd, Tokyo, Japan). The isolates were further examined by using colony PCR for the presence of ompW and toxR genes specific for V. cholerae spp. as described (9 (link)).

Bacteria were isolated from gilthead sea bream (Sparus aurata L.) and shellfish (Penaeus indicus H. Milne-Edwards). These samples were obtained from a local market in Hail region-Saudi on 25 February 2022. Fish with red spots on their skin were targeted, as this is an indication of microbial infection. Upon arrival, gilthead sea bream and prawns were immediately washed using sterile seawater, gutted, headed, and shucked with a sterile knife. Twenty-five grams from prawn abdomen meat, and the intestines, gills, and muscle meat from S. aurata were enriched in 225 mL of alkaline peptone water supplemented with 1% NaCl [18 (link)]. The inoculated broth media was incubated overnight at 37 °C. After incubation, a loopful from each enrichment culture was steaked onto thiosulfate–citrate–bile salt–sucrose agar (TCBS) (Agar; Sigma Aldrich, Darmstadt, Germany) and onto Vibrio ChromoSelect agar (Sigma Aldrich, Germany), before incubating for 18 to 24 h at 37 °C.

The morphology of strain NIT-SL11 was observed under a field-emission scanning electron microscope (JSM-7800F; JEOL Ltd., Tokyo, Japan) operating at 1.0 kV [19 (link)], and its spore-forming ability and Gram-stainability were checked using optical microscopy, as previously described [24 (link)]. The salinity, temperature, and pH tolerance of NIT-SL11 were evaluated by measuring the growth of cells in DHB-CO₃-AF medium. Salinity tolerance was examined by supplementing with 0 to 8% (w/v) of NaCl. The pH of the bicarbonate-free medium was adjusted to the range of 5.2 to 8.6 by the addition of sodium bicarbonate in the medium and CO₂ in the headspace for the pH tolerance tests. The cell growth at different temperatures was tested in the range between 4 °C and 10 to 40 °C, with approximately 5 °C intervals.
The ability of strain NIT-SL11 to utilize an e⁻ donor was determined by observing cell growth with the following substances in combination with reduction of 5 mM fumarate: 10 mM of formate, acetate, butyrate, lactate, pyruvate, succinate, propionate, malate, isobutyrate, caproate, benzoate, phenol, methanol, isopropanol, ethanol, butanol, glucose, fructose, and glycerol, and 0.5 g/L of peptone and yeast extract. Potential electron acceptors used by strain NIT-SL11 were assayed by observing cell growth in 10 mM each of fumarate, malate, sulfate, and thiosulfate; 5 mM of AQDS and nitrate; and 20 g/L of elemental sulfur with oxidation of 5 mM acetate. The production of electric current by the strain NIT-SL11 was evaluated via electrochemical cultivation using a graphite plate inoculated with NIT-SL11, as previously described [12 (link)].